SFRP1 promoter methylation and expression in human trabecular meshwork cells.
SFRP1 promoter methylation and expression in human trabecular meshwork cells.
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DOI：
10.1016/j.exer.2012.01.003
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发表时间：
2012-04
影响因子：
3.4
通讯作者：
Clark AF
中科院分区：
文献类型：
--
作者：
Mao W;Rubin JS;Anoruo N;Wordinger RJ;Clark AF
青光眼是世界范围内导致失明的主要原因。在原发性开角型青光眼（POAG）患者中，小梁网（TM）功能受损导致眼内压（IOP）升高，这是发生视神经病变的主要危险因素。我们的前期研究表明，Wnt信号通路组分在人TM（HTM）中表达，并且Wnt抑制剂分泌型卷曲相关蛋白1（SFRP 1）在肿瘤TM（GTM）中升高。升高的SFRP 1增加小鼠眼睛和灌注培养的人眼前段的IOP。然而，GTM中SFRP 1升高的原因仍然未知。启动子甲基化在调节某些癌细胞中SFRP 1表达中起关键作用。鉴于此，我们研究了启动子甲基化是否也参与了TM中SFRP 1的差异表达。两种正常TM（NTM）和两种GTM细胞株在汇合后再培养7天。同时分离RNA和基因组DNA（gDNA），通过定量PCR（qPCR）比较SFRP 1表达水平，并通过亚硫酸氢盐转化和甲基化敏感性高分辨率熔解分析（MS-HRM）确定SFRP 1启动子甲基化状态。为了研究DNA甲基化抑制剂是否影响TM细胞中SFRP 1的表达，用2 μM 5-氮杂-2 ′-脱氧胞苷（AZA-dC）或不加AZA-dC处理4天。分离RNA以通过qPCR比较SFRP 1表达。此外，使用人癌细胞系NCI-H460作为阳性对照。我们发现两种GTM细胞株的SFRP 1表达水平显著高于两种NTM细胞株。然而，所有四种TM细胞株的SFRP 1启动子均未甲基化。此外，AZA-dC处理不影响任何TM细胞株中的SFRP 1表达（n = 3，p > 0.05）。相反，NCI-H460细胞的高甲基化SFRP 1启动子被相同的处理部分去甲基化。AZA-dC处理还使NCI-H460细胞中的SFRP 1表达升高约两倍（n = 3，p < 0.01）。我们的数据表明，SFRP 1在HTM细胞中的差异表达不是由于差异启动子甲基化。
Glaucoma is a leading cause of blindness worldwide. In primary open angle glaucoma (POAG) patients, impaired trabecular meshwork (TM) function results in elevated intraocular pressure (IOP), which is the primary risk factor of developing optic neuropathy. Our previous studies showed that Wnt signaling pathway components are expressed in the human TM (HTM), and the Wnt inhibitor, secreted frizzled-related protein 1 (SFRP1) is elevated in the glaucomatous TM (GTM). Elevated SFRP1 increased IOP in mice eyes and in perfusion cultured anterior segments of the human eye. However, the cause of elevated SFRP1 in the GTM remains unknown. Promoter methylation plays a key role in regulating SFRP1 expression in certain cancer cells. In light of this, we studied whether promoter methylation is also involved in SFRP1 differential expression in the TM. Two normal TM (NTM) and two GTM cell strains were cultured for an additional 7 days after they were confluent. RNA and genomic DNA (gDNA) were isolated simultaneously to compare SFRP1 expression levels by quantitative PCR (qPCR) and to determine SFRP1 promoter methylation status by bisulfite conversion and methylation-sensitive high resolution melting analysis (MS-HRM). To study whether DNA methylation inhibitors affect SFRP1 expression in TM cells, the four TM cell strains were treated with or without 2 μM 5-aza-2′-deoxycytidine (AZA-dC) for 4 days. RNA was isolated to compare SFRP1 expression by qPCR. In addition, a human cancer cell line, NCI-H460, was used as a positive control. We found that the two GTM cell strains had significantly higher expression levels of SFRP1 than the two NTM cell strains. However, the SFRP1 promoter of all four TM cell strains was unmethylated. In addition, AZA-dC treatment did not affect SFRP1 expression in any of the TM cell strains (n = 3, p > 0.05). In contrast, the hypermethylated SFRP1 promoter of NCI-H460 cells was partially demethylated by the same treatment. AZA-dC treatment also elevated SFRP1 expression by approximately two fold in NCI-H460 cells (n = 3, p < 0.01). Our data suggest that the differential expression of SFRP1 in HTM cells is not due to differential promoter methylation.
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DOI：
10.1007/s004170000252
发表时间：
2001-03-01
影响因子：
2.7
作者：
Picht, G;Welge-Luessen, U;Lütjen-Drecoll, E
通讯作者：
Lütjen-Drecoll, E
影响因子：
8
作者：
Nojima, M.;Suzuki, H.;Shinomura, Y.
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Shinomura, Y.
DOI：
10.1007/s004170000241
发表时间：
2001-02-01
影响因子：
2.7
作者：
Inatani, M;Tanihara, H;Honda, Y
通讯作者：
Honda, Y
DOI：
10.1073/pnas.94.13.6770
发表时间：
1997-06-24
影响因子：
11.1
作者：
Finch, PW;He, X;Rubin, JS
通讯作者：
Rubin, JS
影响因子：
4.7
作者：
Licchesi, Julien D. F.;Westra, William H.;Herman, James G.
通讯作者：
Herman, James G.